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Regulation of 3D organization by CTCF

Jian-Feng Xiang and Victor G Corces

Studies of nuclear architecture using multivalent proteins [1]. The use of high throughput chro-

conformation capture methods have provided a detailed view mosome conformation capture methods has resulted in a

of how chromatin folds in the 3D nuclear space. New variants of more detailed view of chromatin 3D organization. Based on

this technology now afford unprecedented resolution and allow the original results in which the resolution of the Hi-C data

the identification of ever smaller folding domains that offer new ranged between 1 Mb and 50 kb, the general consensus in

insights into the mechanisms by which this organization is the field suggests that mammalian are orga-

established and maintained. Here we review recent results in nized into large >1 Mb compartments, identified by PC1

this rapidly evolving field with an emphasis on CTCF function, after Principal Component Analysis (PCA), that can be

with the goal of gaining a mechanistic understanding of the classified into two types, A and B, which respectively

principles by which chromatin is folded in the eukaryotic correlate with active or inactive chromatin [2]. In addition

nucleus. to large compartments, chromosomes contain smaller

Topologically Associating Domains (TADs) that can be

Address identified using algorithms that detect changes in the

Emory University School of Medicine, Department of Human Genetics,

directionality of interactions. A subset of TADs contains

615 Michael Street, Atlanta, GA 30322, USA

CTCF at the borders and correspond to CTCF loops

whereas others are flanked by actively transcribed genes

Corresponding author:

Corces, Victor G ([email protected], [email protected]) with or without CTCF [3]. Here we use the term loop to

refer to a domain created by relatively stable point to point

contacts, such as those mediated by CTCF/cohesin, and

Current Opinion in Genetics and Development 2021, 67:33–40

visible in Hi-C heatmaps as strong punctate signal at the

This review comes from a themed issue on Genome architecture and

summit of the domain. Loops are different from other

expression

contact domains formed by interactions among sequences

Edited by Susan Gasser and Gerd Blobel

within the domain mediated by proteins present in these

sequences and corresponding to their transcriptional state.

In this review we first consider the distinct contributions of

transcriptional state and CTCF/cohesin to 3D chromatin

https://doi.org/10.1016/j.gde.2020.10.005 organization. We then focus on mechanisms by which

0959-437X/ã 2020 Elsevier Ltd. All rights reserved. CTCF function is regulated to promote the formation of

stable loops that can modulate enhancer–promoter

communication.

3D nuclear organization, transcription, and

CTCF loops

Introduction Results from high resolution Hi-C, around 1 kb for mam-

The eukaryotic genome is organized in the three- malian cells and 250 bp for Drosophila, suggest that eukary-

dimensional nuclear space in a manner that responds to otic genomes are organized into small contact domains

and facilitates the regulation of nuclear processes. This containing one or several closely spaced genes in the same

organization therefore affects, and may be affected by, transcriptional state. Since these contact domains can be

critical nuclear functions such as transcription, replication, identified by PCA using bin sizes of 5À10 kb, they have

recombination, and DNA repair. Until recently, insights been called compartment(al) domains to highlight their

into the 3D organization of the genetic material have come similarity to the original >1 Mb compartments [4,5]. Inter-

mostly from the use of microscopy, which has given us a actions among compartmental domains give rise to the

broad view of nuclear architecture. Results from these plaid pattern observed in Hi-C heatmaps. This interaction

studies suggest that actively transcribed regions interact pattern explains the formation of membraneless organelles

to form various types of membraneless organelles such as or biomolecular condensates observed by microscopy and

transcription factories, regions of the genome containing biochemical studies. Recent analyses of chromatin archi-

H3K27me3 and Polycomb (Pc) complexes PRC1 and tecture using variations of the original Hi-C method have

PRC2 congregate at Pc bodies, and regions containing shed further light into the existence of small contact

H3K9me3 and HP1 such as come together domains that correlate with transcriptional state and may

to form chromocenters. Biochemical analyses of proteins represent the basic unit of chromosome organization.

involved in these contacts suggest that these structures Micro-C employs micrococcal nuclease, rather than restric-

are biomolecular condens formed by interactions among tion enzymes, to digest the chromatin, thus allowing a

www.sciencedirect.com Current Opinion in Genetics & Development 2021, 67:33–40

34 Genome architecture and expression

uniform nucleosome-size length of the digested fragments domains alternate with non-transcribed sequences, which

[6,7]. Micro-C allows the visualization of compartmental may contain H3K27me3, H3K9me3, or neither, and form

domains composed of single genes, including enhancer– self-interacting compartmental domains. Each of these

promoter interactions, and involved in long-range interac- types of domains interact with other domains in the same

tions with other compartmental domains. When expressed, transcriptional state to give rise to the plaid pattern

these domains are disrupted by inhibition of transcription, observed in Hi-C heatmaps away from the diagonal. These

suggesting that they arise as a consequence of interactions long-range interactions are heterogeneous in different cells

among proteins involved in the transcription process [6,7], of a population. Therefore, this organization is a conse-

probably including initiation, splicing and termination. quence of the transcriptional state of sequences that is, an

This may explain why simply depleting the different emergent property of the one-dimensional epigenetic

RNA polymerases has little effect on chromatin 3D orga- information present in the chromatin fiber [12]. However,

nization [8], sinceit is possiblethat other components of the once this organization is established, it can affect gene

transcription machinery or proteins associated with cova- expression by increasing the concentration of various fac-

lent histone modifications may still remain on chromatin, tors in biomolecular condensates established in distinct

although this has not been confirmed experimentally. nuclear compartments.

Small contact domains that correlate with transcriptional

state have also been observed using CAP-C, a derivative of The regulation of the transcriptional state-dependent 3D

Hi-C that uses multifunctional chemical crosslinkers to organization of the genome takes place by well-estab-

capture long-range chromatin interactions [9]. CAP-C lished biochemical principles determined by the affinities

achieves much higher resolution, allowing the identifica- of transcription factors for specific DNA sequences and

tion of short-range interactions, which are present close to for other proteins, which allow the recruitment of protein

thediagonal ofHi-Cheatmapsandnotresolvedbystandard complexes to chromatin. Superimposed on this transcrip-

Hi-C. Using CAP-C, it has been possible to characterize tional state-dependent aspect of nuclear architecture is

loop domains that is, contact domains with strong punctate the phenomenon of cohesin extrusion, which takes place

signal at the summit and identifiable by tools such as throughout the genome and can be stopped by CTCF.

HiCCUPS. Loop domainsareformed by cohesin extrusion, Interactions within and between compartmental domains

have convergent CTCF sites at their anchors, and are are disrupted by CTCF and cohesin mediated loop

affected by CTCF depletion. Nonloop domains contain extrusion [12]. The cohesin complex is loaded throughout

single genes,rangeinsize between 10 kb and40 kb, andare the genome, or perhaps preferentially at NIPBL sites.

affected by transcription inhibition when present in the A Cohesin extrusion may be slowed down at genomic sites

compartment [9]. The independent contribution of CTCF containing specific proteins or large protein complexes

and transcription to 3D chromatin organization has been [6,7]. However, cohesin extrusion is stopped by sites

recently tested in a series of elegant experiments involving bound by the CTCF protein, preferentially when the

either swapping the Xist/Tsix transcriptional units [10] or sites are arranged in a convergent orientation [13–15].

inserting a 2 kb sequence containing a CTCF site and a This retention of the cohesin complex at CTCF sites

TSS [11]. The consequence of the presence of this 2 kb results in the formation of relatively stable loops that can

fragment in different genomic locations was analyzed by be visualized in Hi-C heatmaps as strong puncta that

Hi-C. Depending on the genomic context, the inserted represent interactions between two anchors containing

CTCF site can make loops with adjacent sites in the CTCF. The strong punctate signal clearly visible above

genome whereas the TSS creates new domains by forming the surrounding background in Hi-C heatmaps suggests

directionalcontactswith downstreamsequences.When the that these loops may be sufficiently stable to be simulta-

fragment is inserted in B compartment sequences, this neously present in a majority of cells in a population [16].

TSS-dependent domain is able to form a new A compart- The formation of these stable loops favors interactions

ment and establishes long-range contacts with other A- between sequences inside of the loop and precludes

compartment domains, suggesting that domains induced interactions between sequences inside and outside of

by expression from the inserted TSS correspond to what we the loop, thus having the potential of regulating interac-

have referred to as compartmental domains. Deletion of tions among regulatory sequences in the genome. There-

CTCF or the TSS independently affect each type of fore, a detailed understanding of the mechanisms by

domain. These results support previous observations in which CTCF/cohesin function can be regulated will be

Drosophila suggesting that the basic units of chromosome critical to understand this aspect of 3D chromatin

organization are small compartmental domains that corre- organization.

spond to the transcriptional state of their sequences. When

present in active chromatin, whether actively transcribing Regulation of CTCF function by DNA

or paused, these domains correspond to single genes,whose methylation

sequences self-interact to form a gene loop. Adjacent, CTCF is a ubiquitously expressed and highly conserved

consecutively arranged active genes interact with each protein containing eleven zinc fingers (ZFs). CTCF

other to form larger compartmental domains. These binds to an approximately 41 bp sequence containing

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Regulation of CTCF function Xiang and Corces 35

four modules. A highly conserved 15 bp central core fork during S phase and before histone octamers have

containing modules 2 and 3 is flanked by modules been deposited on nascent chromatin. Interestingly,

1 and 4, which are much less conserved [17]. Only 6% CTCF sites are flanked by hemimethylated DNA at

of all CTCF sites in the genome contain all 4 modules, the DNA entry site into the nucleosome. Although this

and 38% contain the core plus either module 1 or 4. The hemimethylated DNA does not appear to be required for

rest of CTCF genomic sites, around 66%, only contain the DNA binding, its loss affects the ability of CTCF sites to

core motif [17]. This variability in the sequence of CTCF form loops with other sites in the genome [23]. The

binding sites is interesting in the context of results presence of CTCF at unmethylated sites is necessary

describing the structure of the CTCF ZF domain to maintain their unmethylated state. Downregulation of

revealed by X-ray crystallography [18,19]. Each ZFs CTCF, or loss of one copy of CTCF in breast and prostate

3–7 makes contacts with three bases in the DNA major tumors [24], results in methylation of CTCF sites, which

groove of the core consensus [19], while ZF8 acts as a then may further interfere with binding of this protein.

flexible spacer element to allow ZFs 9–11 to bind to This effect should be limited to those CTCF sites in the

module 1 in regions of the genome containing this genome containing CpG at position 2 in the core motif,

sequence [18,19]. These results suggest that ZFs 1–2 which would limit the effect of CTCF copy number loss

and 9–11 may not be required for sequence-specific to the expression of a subset of genes proximal to these

binding at most sites in the genome, opening the class of CTCF sites. This may explain the specific phe-

possibility that these ZFs may have alternative roles in notypes caused by loss of one copy of CTCF in humans,

CTCF function. which include autism, intellectual disability, and cleft

palate [28,29].

Approximately 30% of CTCF-bound sites are different

among cell lines derived from different tissues [20], Regulation of CTCF function by covalent

suggesting that the localization of CTCF in the genome modifications

may partially change during cell differentiation. In Binding of CTCF to chromatin is also affected by several

addition, it has been shown that CTCF occupancy can post-translational covalent modifications. Multiple sites

change in response to environmental stimuli, for example in CTCF can be modified by poly(ADP-ribosyl)ation

temperature or environmental contaminants such as chro- (PARylation), SUMOylation and phosphorylation

mium [21]. It is thus possible that alterations of CTCF [30–33]. PARylated CTCF was first shown to be present

binding play a role in regulating enhancer–promoter in the maternal H19 imprinted control region but also

interactions during the establishment of specific cell found in other parts of the genome. Inhibition of PARP-1

fates. This may explain tissue-specific phenotypes interferes with the ability of CTCF to restrict enhancer–

observed when CTCF expression is altered during devel- promoter interactions [32]. These initial studies agree

opment. The mechanisms by which CTCF is recruited to with results indicating an enrichment of PARylated

specific sites in the genome during cell differentiation are CTCF in cells undergoing cell cycle arrest, which corre-

not clear. A subset of CTCF sites contain CpGs and, lates with loss of CTCF at most sites in the genome [34].

therefore, CTCF binding may be susceptible to the Contrary to these results, PARP-1 stabilizes CTCF bind-

methylation state of its target sequence. In vitro binding ing in the Epstein Barr virus genome [35]. Interestingly,

experiments indicate that cytosine methylation at posi- PARylation of CTCF is metabolically controlled by the

tion 2 of the core motif interferes with CTCF binding b-NAD + salvage pathway, pointing to possible regula-

whereas methylation of the cytosine at position 12 has no tory events to control CTCF PARylation in cancer or

effect [19], suggesting that CTCF would preferentially development. In Drosophila, where CTCF is also PARy-

bind to sites unmethylated in the CpG at position 2 but lated, this modification is required to facilitate interac-

still bind to sites containing 5mC at position 12. Site tions between distant sites [36]. We suggest that the role

specific alteration of DNA methylation by precise geno- of PARylation in the regulation of CTCF function merits

mic editing [22] or genome-wide disruption by depleting further research, especially in the context of findings

DNA methyltransferases [23] or dioxygenases [24] leads indicating that ATP is generated in the nucleus from

to changes in the CTCF binding pattern. In addition to poly(ADP-ribose) [37] and the requirement of ATP for

directly inhibiting the interaction between CTCF and its cohesin extrusion [38].

binding site, the presence of DNA methylation also

blocks CTCF binding by re-positioning nucleosomes SUMOylation of architectural proteins has been shown to

[24]. At its binding sites, CTCF is in equilibrium with affect their function in Drosophila [39] but the role of

a fragile nucleosome containing H3.3 and H2A.Z [25,26], CTCF SUMOylation in controlling its interaction with

and it is flanked by approximately 10 well-positioned chromatin or its ability to form loops has not been

nucleosomes on each side [27]. It is likely that these explored in detail in mammals. There are widespread

positioned nucleosomes are a consequence, rather than a changes in SUMOylation of CTCF sites during the heat

determinant, of CTCF binding. CTCF can probably bind shock response in human cells, and CTCF becomes de-

to DNA immediately after the passage of the replication SUMOylated during hypoxic stress, but the consequence

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36 Genome architecture and expression

of these changes on the role of CTCF in chromatin 3D unclear whether differential expression of the CTCF

organization have not been explored [31,40]. short isoform during development or in response to

stimuli represents a strategy used by cells to regulate

The role of phosphorylation in CTCF function has been CTCF function. A similar role may be played by the

analyzed in more detail than that of other covalent CTCF homologue CTCFL, which is transiently

modifications. Phosphorylated forms of CTCF are pres- expressed during germline development but at very

ent during interphase and mitosis, suggesting that this low levels in normal somatic tissues. CTCFL binds to

modification may play different roles in CTCF function a very similar sequence than CTCF and, if expressed at

during the cell cycle. Early studies identified CK2 as a higher levels, as it does in many cancers, could compete

kinase able to phosphorylate S612 [41] but this initial with CTCF for binding at a subset of sites [51,52]. Since

work should be reanalyzed in the context of our current CTCFL lacks the domain required for cohesin interac-

understanding of how CTCF affects gene expression. tion, this protein is unable to stop cohesin extrusion and to

Phosphorylation of CTCF at T374 and S402, located form stable loops between distant sites in the genome

in the linker space between the central ZFs, by LATS1 [53].

kinase interferes with CTCF binding to chromatin, which

in turns affects expression of a specific subset of genes Several different proteins have been shown to co-localize

[42,43]. This kinase is normally cytoplasmic but translo- with CTCF but their involvement in the regulation of

cates to the nucleus under glucose starvation and may do CTCF function has not been studied in detail until

so under other conditions. Since LATS kinases are central recently. For example, AP-1 has been shown to be

components of the Hippo pathway, it will be interesting recruited to CTCF sites involved in gained interactions

to explore whether signaling through this pathway is responsible for changes in gene expression during mac-

responsible for changes in the binding of CTCF to rophage development and in the response of hESCs to

DNA during cell differentiation. Multiple Thr and Ser temperature stress [21,54]. A subset of 153

residues in CTCF are phosphorylated during mitosis (NUP153) sites in mESCs are present within 5 kb of

[43,44]. Mitotic chromosomes have a unique structure CTCF sites and depletion of NUP153 leads to the loss

determined by loops and, therefore, the for- of CTCF and cohesin binding to chromatin [55]. Given

mation of this structure requires unraveling the standard the genomic distance between the location of these

interphase chromosome organization determined in large proteins, it is unclear whether the effect of NUP153 is

part by cohesin and CTCF [45]. Depending on the cell direct or indirect. As described above, CTCF is in equi-

type, CTCF is present at low to very low levels in librium with a fragile nucleosome at its binding sites, and

metaphase chromosomes [46–48], suggesting that regula- surrounded by 10 well-positioned nucleosomes on each

tion of CTCF binding by phosphorylation may represent side. It is unclear whether CTCF binding requires posi-

a mechanism to ensure depletion of this protein in mitotic tioned nucleosomes flanking its binding site or, once

chromatin. This takes place at T289, T317, T346, T374, bound, CTCF positions the flanking nucleosomes.

S402, S461, and T518, all residues located in the linker Recent results suggest that binding of CTCF requires

region of different ZFs [42,43]. Phosphorylation of CTCF the ISWI complex, and depletion of the SNF2H compo-

at S224 by Polo-like kinase 1 during the G2/M transition nent of ISWI results in loss of CTCF from chromatin [56].

affects the expression of hundreds of genes without In addition to facilitating CTCF binding, some proteins

affecting mitosis or chromatin organization [43,44]. This regulate CTCF function by interfering with its recruit-

Ser residue is located in the amino-terminal domain of ment to DNA. An example is the ChAHP complex, which

CTCF, immediately adjacent to the YDF motif that is composed of CHD4, ADNP, and HP1 [54]. ADNP is a

interacts with cohesin, and it would be interesting to ZF protein that recruits the ChAHP complex to specific

study whether cohesin extrusion is affected by changes sites in the genome to repress expression of genes

in phosphorylation levels of S224. involved in lineage commitment by mechanisms that

do not involve H3K9me3. The ChAHP complex com-

Regulation of CTCF function by interaction petes with CTCF for binding to a subset of genomic sites.

with other proteins Since ChAHP is unable to stop cohesin extrusion, the

Although most studies to date have focused on the presence of this complex interferes with the formation of

analysis of the canonical CTCF protein, human cells also a subset of CTCF loops [57]. Interestingly, a role in

produce other isoforms whose roles have not been facilitating loop formation has also been proposed for

explored in detail. In particular, a short CTCF isoform the ADNP protein, although in this case it is unclear

is encoded by an alternatively spliced transcript lacking whether ADNP participates with the rest of the ChAHP

exons 3 and 4 [49]. This isoforms lacks the N-terminal complex. Another protein that cooperates with CTCF in

region responsible for interactions with cohesin and ZFs the formation of loops is TFIIIC, which is recruited to Alu

1–2 [49]. CTCF genomic sites containing the CTCF elements located near cell cycle genes in response to

short isoform fail to stop cohesin extrusion, leading to serum starvation. TFIIIC then interacts with CTCF

genome-wide decrease in cohesin occupancy [49,50]. It is located at cell cycle gene promoters and these existing

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Regulation of CTCF function Xiang and Corces 37

loops rapidly activate gene expression upon serum expo- Depletion of either CTCF or the cohesin subunit

sure [58]. Rad21 eliminates CTCF loops from the genome based

on Hi-C analyses [4,72]. Recent results illustrate the

Regulation of CTCF function by RNA structural basis for the CTCF–cohesin interaction [50].

CTCF not only interacts with DNA and other proteins, A region in the N-terminus of CTCF containing a YDF

but also binds RNA and this interaction is important to motif interacts with a pocket formed by the SA2-SCC1

regulate CTCF function [59,60]. In fact, the affinity of subunits of cohesin. Interestingly, a similar sequence is

CTCF for RNA is an order of magnitude greater than for present in the cohesin release factor WAPL, suggesting

DNA. However, CTCF does not bind specific RNAs. that perhaps the role of the CTCF–cohesin interaction is

Instead, the RNA interactome of CTCF is composed of to prevent unloading of cohesin by WAPL [50]. As

thousands of transcripts from genes located in close predicted by this model, CTCF mutants lacking the

proximity to CTCF binding sites [59,60]. Interactions N-terminus including the YDF motif cannot form loops

between CTCF and RNA take place through ZF1, ZF10, [73,74]. The requirement for such a precise interaction

ZF11, and 38 amino acids in the C-terminus following the between two very specific and relatively small domains

ZF domain [61,62]. As described above, these ZFs are not have led to the suggestion that stopping cohesin extrusion

involved in the recognition of the core CTCF motif. by CTCF should require additional mechanisms to slow

Deletion of the RNA-binding domains of CTCF results down the movement of the cohesin complex [75]. The

in decreased binding to DNA and cohesin at a subset of flanking arrays of positioned nucleosomes, the formation

genomic regions and a decrease in looping interactions of an unusual DNA structure as a consequence of DNA

mediated by these anchors [61,62]. Since RNA is required binding by CTCF, association with RNA, covalent

to mediate CTCF–CTCF interactions, it has been sug- modifications by bulky SUMO or poly-ADP-ribose, or

gested that the role of RNA at CTCF sites in the genome formation of CTCF aggregates have all been suggested as

is to promote the formation of large CTCF aggregates candidates to pause cohesin extrusion and allow specific

that can help slow down cohesin extrusion to ensure interactions between the CTCF and SA2-SCC1 binding

orientation-dependent CTCF–cohesin interactions (see interfaces [75]. In this context, it is interesting that

below). However, it is unclear why deletion of the RNA Top2B interacts with CTCF and localizes to genomic

binding domain does not affect all genomic sites involved sites for this protein such that the order at loop anchors is

in loop formation. The specific nature of the RNAs Top2B–CTCF–cohesin that is, Top2B is outside of the

involved in this process or the basis for selectivity are loop whereas cohesin is inside [76]. Although there is no

not known. Recently developed methods such as RD- evidence for cohesin extrusion causing DNA supercoil-

SPRITE [63] to simultaneously measure all contacts ing, condensin is capable of introducing positive super-

among RNA and DNA combined with an immunopre- coils into DNA in an ATP-dependent manner [77,78].

cipitation step using antibodies to CTCF should be able While speculative at this time, it may be possible that

to address these questions. It is tempting to speculate that supercoils resulting from transcription [79] or cohesin

RNA modifications might contribute to the specificity of extrusion accumulate at the inside of loop anchors, and

CTCF–RNA interactions in view of results showing that that this contributes to slowing cohesin extrusion to allow

6

m A modification of RNA can regulate chromatin acces- it to interact with CTCF.

sibility [64].

Cohesin is a large ring-shaped protein complex with

Regulation of CTCF function by control of several alternative subunits subject to covalent mod-

cohesin extrusion ifications, both of which can serve as regulatory steps

An important role of CTCF appears to be to stop cohesin of cohesin extrusion during interphase to control the

extrusion. In doing so, CTCF makes anchors of loops that type and stability of loops. In addition to the core

have one of two functions — they either insulate genes SMC3, SMC1A, and RAD21 subunits, the cohesin

and regulatory sequences inside the loop from those complex present in somatic cells contains one of

located outside or they tether regulatory sequences to two STAG subunits, STAG1 and STAG2. Cohesin

their cognate promoters. Cohesin loads either at NIPBL complexes containing either of these two subunits

sites or randomly throughout the sequences that eventu- have overlapping but also distinct binding locations

ally will form the loop. Cohesin will then extrude a loop on chromatin and they form different sized loops

until it encounters two CTCF sites arranged in a conver- [80,81]. STAG1 can be modified by the acetyltransfer-

gent head-to-head orientation, a phenomenon that is ase ESCO1, and this acetylation protects cohesin from

supported by computational modelling [13,65] and in release by WAPL. As a consequence, acetylated

vitro single molecule studies [66–68]. Loop extrusion is STAG1-containing loops are longer and stable for

an evolutionarily conserved mechanism by which cells periods of several hours while those formed by SATG2

segregate interphase and mitotic chromatin, a conclusion are shorter and stable for minutes [82]. This may be

supported by evidence in Bacillus subtilis [69], yeast [70], important during cell differentiation when stabiliza-

Xenopus egg [71], mouse [72] and human cells [68]. tion of loops for long period of times may allow cells in

www.sciencedirect.com Current Opinion in Genetics & Development 2021, 67:33–40

38 Genome architecture and expression

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Conflict of interest statement

Acad Sci U S A 2015, 112:E6456-E6465.

Nothing declared.

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content is solely the responsibility of the authors and does not necessarily

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